Nervous system development encompasses generation of neural cells, followed by their positioning and differentiation to culminate with establishment of specific connections that enable the execution of innumerable functions. Although both the developing and mature nervous system exhibit tremendous plasticity, comparatively, the former has a greater capacity for remodeling. When the neural tissue is injured or its function is imbalanced, as for patients suffering from spinal cord injury or epilepsy, recreating the remarkable plasticity of the developing nervous system towards repairing and regenerating damaged cells and connections is the ultimate goal. Understanding developmental processes becomes crucial to devising therapies targeting nervous system regeneration and repair. Two major developmental cues are key for nervous system development, the morphogentic protein, Sonic hedgehog (Shh), and early electrical activity. Although many aspects of their action are known, there has been no previous consideration of the interaction between them. This research project wil study the molecular mechanisms underlying the interplay between Shh and electrical activity and how their interaction affects nervous system development and maturation. This study will test the hypothesis that the Shh gradient across the dorsoventral axis of the nervous system contributes to establish a gradient of calcium-dependent electrical activity across the dorsoventral developing spinal cord that in turn regulates neuronal differentiation. Pharmacological and molecular manipulations of Shh gradient and signaling wil be implemented in developing Xenopus embryos. Calcium dynamics will be imaged in neurons on the dorsal and ventral surfaces of the neural tube of control and experimentally perturbed embryos. Reciprocally, manipulations of calcium spike activity will be carried out and the consequences to Shh signaling in the developing spinal cord will be assessed. To investigate the functional consequences of the interplay between Shh signaling and calcium spike activity, a crucial biological outcome will be studied: dorsoventral differentiation of developing spinal neurons. The involvement of calcium spike activity-dependent pathways in this process will be studied in vivo following pharmacological and molecular perturbations of Shh signaling and electrical activity. This project may lead to new ways of thinking of how the nervous system develops and how different pathways interact to promote its formation and maturation. When generation and differentiation of neural cells is needed to reestablish lost connections, the ability to direct the affected system toward the production of appropriate number and type of cells is paramount. This investigation of mechanisms underlying neuronal differentiation may set the basis for devising treatments for neurological disorders such as pediatric epilepsy and spinal cord injury, in which both the reestablishment of balanced excitability and the reposition of damaged cells are key events for promoting recovery. PUBLIC HEALTH RELEVANCE: This project investigates the complex developmental process of neuronal specification and differentiation during nervous system development. We will elucidate the interaction between two major developmental cues, electrical activity and the morphogenetic protein Sonic hedgehog. When the neural tissue is injured or its function is imbalanced, as for patients suffering from spinal cord injury or epilepsy, recreating the remarkable plasticity of the developing nervous system towards repairing and regenerating damaged cells and connections is the ultimate goal. The knowledge of how different patterns of activity are established during development and how they relate to other regulatory factors will help devising therapies aimed at inducing restorative plasticity in a pathological context of loss or impaired synaptic connectivity. This study wil contribute to the understanding of how to direct the affected system toward the production of appropriate number and type of cells when generation and differentiation of neural cells are needed. Moreover, results from this project may identify novel non-canonical mechanisms of action of Sonic hedgehog that may be of relevance to many fields of research, including developmental biology, neuroscience, cancer, stem cells and tissue repair.